Field of the Invention
The present application relates generally to NF-κB subunit c-Rel and c-Rel activation through O-GlcNAcylation. Methods for identifying compounds that regulate O-GlcNAcylation of c-Rel and methods for identifying compounds that block O-GlcNAcylation of c-Rel are provided.
Description of the Related Art
NF-κB is a pleiotropic, evolutionarily conserved transcription factor family, having multiple roles in cell survival, development, apoptosis, immunity and inflammatory responses. The NF-κB family is composed of five members that function as dimers: p65 (RelA), RelB, c-Rel, p50 (and its precursor p105) and p52 (and its precursor p100). NF-κB members are preformed proteins that reside, in basal state, mostly in the cytoplasm bound to inhibitory proteins of the IκB family. In general, activation occurs through a signal-induced phosphorylation and proteasome-mediated degradation of IκB. This releases the previously bound NF-κB, which translocates to the nucleus and activates transcription.
Because the NF-κB members are preformed proteins, their initial activation and activity is mainly regulated by post-translational modifications rather than induction of their synthesis. O-GlcNAcylation is a form of intracellular post-translational protein modification. It involves attachment of the monosaccharide N-acetylglucosamine to serine and threonine residues (O-GlcNAc), in nuclear and cytoplasmic proteins in multicellular eukaryotes. O-GlcNAcylation is a reversible process catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) mediating addition and removal of O-GlcNAc, respectively, giving rise to functional diversity of proteins. O-GlcNAcylation levels play an important role in many key cellular processes, and O-GlcNAcylation is essential for T cell function.
O-GlcNAcylation can be increased under hyperglycemic conditions and enhance NF-κB-dependent transcription. Both O-GlcNAcylation and NF-κB activation have been associated with experimental and clinical diabetes. Growing evidence supports a pivotal role for O-GlcNAcylation in the activation of NF-κB in B and T cells (Golks et al. Embo J 26:4368-4379 (2007)). Recently, regulation of NF-κB p65 by O-GlcNAc glycosylation has received some attention with the identification of the sites of modification and their role in influencing p65 function (Yang et al. Proc Natl Acad Sci USA 105:17345-17350 (2008); Allison et al. Proc Natl Acad Sci USA 109:16888-16893 (2012)).
Although both NF-κB and O-GlcNAcylation were discovered some 25 years ago, the role of this post-translational modification in regulating NF-κB subunits other than p65, remain largely uncharacterized. As discussed in detail below, it has now been discovered that O-GlcNAcylation of c-Rel is necessary for DNA binding and transactivation following T-Cell Receptor stimulation. Thus, inhibiting O-GlcNAcylation of c-Rel provides an effective way to decrease cytokine production and T cell function and ameliorate autoimmune response in conditions such as type-I diabetes.